Molecules and methods for improved immunodetection of small molecules such as histamine

Abstract

Embodiments of various aspects described herein are directed to methods, compositions, kits for detecting a target molecule in a sample. In particular, there is described herein a multivalent approach which provides an efficient method for detection of small molecules and screening of binding molecules (e.g., antibodies). The multivalent approach uses two or more small molecules in a domain that is attached to a substrate through a linking group. The multivalent domain is free to extend, e.g., into a solution, for presentation to a binding compound such as an antibody.

What is claimed is: 
     
         1 . A compound comprising:
 (i) a substrate binding domain;   (ii) a branching domain comprising a plurality of small molecules each small molecule linked to a branch of a branch-point; and   (iii) a linker linking the substrate binding domain and the branching domain.   
     
     
         2 . The compound of  claim 1 , wherein the small molecules independently have a molecular weight of less than 1,000 Da. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The compound of  claim 1 , wherein the linker has a length between 5 and 200 angstroms. 
     
     
         8 .- 9 . (canceled) 
     
     
         10 . The compound of  claim 1 , wherein the branch-point comprises at least one lysine. 
     
     
         11 . The compound of  claim 10 , wherein at least one small molecule is linked to the alpha-amino group the at least one lysine and at least one small molecule is linked to the epsilon-amino group of the at least one lysine. 
     
     
         12 . The compound of  claim 10 , wherein the branch-point comprises a first lysine linked to a second lysine, and wherein the carboxyl group of the first lysine is linked to the epsilon-amino group of second lysine. 
     
     
         13 . The compound of  claim 10 , wherein the branch-point comprises a first lysine, a second lysine and a third lysine, and wherein the carboxyl group of the first lysine is linked to the epsilon-amino group of the second lysine, and the carboxyl group of the third lysine is linked to the alpha-amino group of the first or second lysine. 
     
     
         14 .- 18 . (canceled) 
     
     
         19 . The compound of  claim 1 , wherein the substrate binding domain comprises a reactive group or one member of a binding pair. 
     
     
         20 .- 22 . (canceled) 
     
     
         23 . The compound of  claim 1 , wherein the branching domain comprises: 
       
         
           
           
               
               
           
         
       
       wherein,
 d+f≥2 (e.g., between about 2 and 100), d≥c, and e≥f, wherein c, d, e and f are integers and each M is a small molecule. 
 
     
     
         24 . (canceled) 
     
     
         25 . The compound of  claim 1 , wherein the branching domain has the formula C(x) d M b ,
 wherein:   C is a sub unit of the branching domain having a maximum of possible x branches, and the branch-point comprises one or more sub unit C and at least one subunit C is attached to the linker through a branch;   M is a small molecule attached to the subunit C through a branch;   a is an integer≥1; and   b is an integer≥2, provided that b≤(a)(x−2)+1.   
     
     
         26 . The compound of  claim 1 , wherein the compound is linked to a substrate via the substrate binding domain. 
     
     
         27 .- 28 . (canceled) 
     
     
         29 . The compound of  claim 26 , wherein a surface of the substrate is coated with a proteinaceous material, wherein the proteinaceous material can optionally be reversibly or non-reversibly denatured and/or cross-linked. 
     
     
         30 . (canceled) 
     
     
         31 . A method for detecting presence of an analyte in a sample, the method comprising:
 (i) contacting a sample suspected of comprising an analyte with a compound of  claim 1 ; and   (ii) detecting binding of an analyte binding molecule to the compound of  claim 1 .   
     
     
         32 . The method of  claim 31 , wherein the analyte binding molecule is an antibody. 
     
     
         33 . The method of  claim 31 , wherein said detecting of step (ii) comprises producing a chromogenic, fluorescence or electrochemical signal. 
     
     
         34 . The method of  claim 31 , wherein the analyte binding molecule comprises a detectable label. 
     
     
         35 . The method of  claim 31 , wherein said detecting step comprises contacting the sample from (i) with a molecule capable of binding with the analyte binding molecule and comprises a detectable label. 
     
     
         36 . The method of  claim 31 , wherein the analyte is histamine or dinitrophenol. 
     
     
         37 . The method of  claim 31 , wherein the molecule of  claim 1  is linked to an electrode surface by the substrate binding domain and the analyte binding molecule includes an electroactive component, and wherein the analyte binding molecule is detected by the electrode when the electroactive component is proximate to the electrode. 
     
     
         38 .- 40 . (canceled) 
     
     
         41 . A method for selecting a ligand capable of binding a small molecule, the method comprising:
 (i) contacting a test ligand with a compound of  claim 1 ; and   (ii) detecting binding of the test ligand with the compound of  claim 1  in the presence and in the absence of the small molecule, and   selecting the test ligand having reduced binding in the presence of the small molecule.   
     
     
         42 .- 48 . (canceled)